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Diagnosis and prognostic stratification of myelodysplastic syndromes (MDS) have been complemented by new techniques, including flow cytometry and NGS. To analyze the relationship between molecular and cytofluorimetric data, we enrolled in this retrospective study, 145 patients, including 106 diagnosed with MDS and 39 controls. At disease onset, immunophenotypic (IF), cytogenetic tests, and cytomorphological (CM) examination on bone marrow were carried out in all patients, while NGS was performed in 58 cases. Ogata score presented a specificity of 100% and a sensitivity of 59%. The detection of at least two phenotypic aberrancies in Ogata negative patients increased the sensitivity to 83% and specificity to 87%. Correlations were identified between IF aberrancies and mutations, including positive Ogata>2 and mutations in SRSF2 (p=0.035), CD15 and U2AF1 (0.032), CD56 and DNMT3A (p=0.042), and CD38 and TP53 (p=0.026). In multivariate analysis, U2AF1 mutations, associated with del(20q) and/or abnormalities of chromosome 7 (group 4 as defined by the EuroMDS score), significantly correlated with an inferior overall survival (p=0.019). These parameters and Ogata score>2 also showed a significant correlation with inferior event-free survival (p=0.023 and p=0.041, respectively). Both CM and FC features correlated with prognosis and mutational patterns. In an integrated MDS work-up, these tools may guide indications for mutational screening for optimal risk stratification., (© 2023. The Author(s).)

Baseline cytogenetic/genetic features have been widely recognized to play a critical prognostic role in acute myeloid leukemia (AML) and have proven useful in designing risk-adapted treatment strategies. Nevertheless, to improve further the outcome of AML patients we are still in need of accurate methods to explore the quality of response and to adequately discriminate patients who are likely to relapse over time from those who are in deep and stable remission. In this view, is it well established that measurement of leukemic cells surviving chemotherapy (called measurable residual disease, MRD) during the course of treatment may be a reliable biomarker in predicting relapse. Detection of MRD relies on highly sensitive techniques, such as quantitative polymerase chain reaction and multiparametric flow cytometry, which, due to their levels of specificity and sensitivity, are increasingly included in the decision-making process of AML treatment. In the present manuscript, we will review the current techniques of MRD investigation and their clinical contribution to AML management.

Incidence and effect on outcome of central nervous system (CNS) involvement in adult patients with acute myeloid leukemia (AML) is not clearly defined. To address this issue, 103 consecutive adult patients with newly diagnosed AML, regardless of neurologic symptoms, were submitted to a routine explorative lumbar puncture. Cerebrospinal fluid (CSF) samples were collected from 65 males and 38 females. All 103 CSF samples were examined by conventional cytology (CC) whereas 95 (92%) also by flow cytometry (FCM). At diagnosis, 70 patients (68%) were CNS negative (CNS-), whereas 33 (32%) were CNS positive (CNS+). In 11 of 33 (33%), CNS infiltration was documented either by CC or FCM , in 21 (67%) only by FCM. CNS positivity was significantly associated with a M4-M5 phenotype of the underlying AML (P = .0003) and with high levels of lactate dehydrogenase (P = .006). Overall, 80 of 103 (78%) achieved complete remission with no significant differences between CNS+ and CNS- patients. Five-year disease-free survival and overall survival were found to be shorter in CNS+ patients than in those CNS- (18% vs 50%, P = .006 and 19% vs 46%, P = .02, respectively). In multivariate analysis, CNS status and age were found to affect independently overall survival. In conclusion, the incidence of CNS involvement in adult patients with newly diagnosed AML is higher than expected. Regardless of neurologic symptoms, it should always be searched at diagnosis; CSF samples should routinely be investigated by FCM since a certain proportion of CNS involvements might remain undetected if examination is exclusively CC based., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Conventional cytology (CC) of cerebrospinal fluid (CSF) fails to demonstrate malignant cells in up to 45 % of patients with acute lymphoblastic leukemia or lymphoblastic lymphoma (ALL/LL) in whom occult leptomeningeal disease is present. Flow cytometry (FCM) is considered more sensitive than CC, but clinical implications of CC negativity/CC positivity are not yet established. CSF samples from 38 adult patients with newly diagnosed ALL/LL were examined. Five (13 %) and nine (24 %) specimens were CC positive-FC positive (FCM(pos)/CC(pos)) and CC negative-FC positive (CC(neg)/FCM(pos)), respectively. The remaining 24 (63 %) samples were double negative (CC(neg)/FCM(neg)) (p = 0.001). CC(neg)/FCM(pos) patients showed a significantly shorter overall survival (OS) compared to CC(neg)/FCM(neg) ones. In multivariate analysis, the status of single FCM positivity was demonstrated to affect independently duration of OS (p = 0.005). In conclusion, FCM significantly improves detection of leptomeningeal occult localization in ALL/LL and appears to anticipate an adverse outcome. Further prospective studies on larger series are needed to confirm this preliminary observation.

The term minimal residual disease (MRD) describes the situation in which, after chemotherapy for acute leukemia (AL), a morphologically normal bone marrow (BM) can still harbor a relevant amount of residual malignant cells. Several techniques are now amenable to investigate MRD, and all together they have designated a new era in which a re-definition of the current criteria of complete remission (CR) is required. Depending upon the measured level of MRD we can distinguish a variety of clinical situations ranging from a potentially cured disease to short-term remission. In the context of this spectrum of conditions there would be room for different therapeutic strategies ranging from no further therapy to pre-emptive therapy to treat early relapses (immunologic and/or molecular relapses). This review will focus on the state of art of MRD detection in acute myeloid leukemia (AML) using multidimensional flow cytometry (MFC), and will cover the laboratory and clinical aspects of this approach.

The absence of measurable residual disease (MRD) in morphologic complete remission (CR) represents a favorable prognosticator in acute myeloid leukemia (AML) [[1]]. At the diagnostic evaluation, patients were defined as LSCs negative (LSC-0) in case of undetectable LSCs count, LSC SP low sp and LSC SP high sp when LSCs were detected below or above a threshold of 0.03%, respectively [[4]]. Indeed, patients with a high LSC burden at diagnosis have an unfavorable outcome unless LSCs are cleared by chemotherapy. [Extracted from the article]

The choice of either induction or post-remission therapy for adults with acute myeloid leukemia is still largely based on the "one size fits all" principle. Moreover, pre-treatment prognostic parameters, especially chromosome and gene abnormalities, may fail in predicting individual patient outcome. Measurement of minimal residual disease (MRD) is nowadays recognized as a potential critical tool to assess the quality of response after chemotherapy and to plan post-remission strategies that are, therefore, driven by the individual risk of relapse. Polymerase chain reaction and multiparametric flow cytometry have become the most popular methods to investigate MRD since they have been established as sensitive and specific enough to allow MRD to be studied serially. In the present review, we will examine the evidence supporting the appropriateness of incorporating MRD detection into the AML risk assessment process. A comprehensive prognostic algorithm, generated by combining pre-treatment cytogenetics/genetics and post-treatment MRD determination, should promote advances in development of personalized therapeutic approaches. [ABSTRACT FROM AUTHOR]

In chronic lymphocytic leukemia (CLL), inhibition of spontaneous apoptosis determines a worse prognosis and increasing evidences show that disease progression relies also upon cycling CLL cells. We investigated bcl-2, as measure of apoptosis, and CD71, as measure of proliferation, by flow cytometry in 265 patients with CLL. Combining bcl-2 with CD71 values, we defined three subgroups: (1) bcl2 − CD71−; (2) bcl2 + CD71+; and (3) bcl2 + CD71− or bcl2− CD71+. Both a shorter progression-free survival (PFS) and overall survival (OS) were observed in ZAP-70+ ( p < 0.00001) and in patients with bcl2 + CD71+ ( p < 0.00001 and p = 0.02). The patients with discordant in bcl2 + CD71− and bcl2− CD71+ showed an intermediate outcome. Noteworthy, patients with bcl2 + CD71+ showed a shorter PFS within ZAP-70 negative subgroup ( p = 0.00009). In multivariate analysis of PFS, age ( p = 0.005), beta-2 microglobulin (B2-M) ( p = 0.003), bcl-2 ( p = 0.004), CD49d ( p = 0.001), and ZAP-70 ( p < 0.001) resulted to be significant prognostic factors. The independent prognostic significance of B2-M ( p = 0.009) and bcl-2 ( p = 0.03) was confirmed within ZAP-70 negative patients. Bcl-2 and CD71 can be considered as interesting progression indicators, which should be validated in an independent cohort of patients, to take timely therapeutic decisions in CLL. [ABSTRACT FROM AUTHOR]

Summary. Concurrent resistance mechanisms, such as P-glycoprotein (PGP) and bcl-2, may contribute to a worse outcome in adult acute lymphoblastic leukaemia (ALL). Between 1990 and 2000, we analysed PGP and bcl-2 by flow cytometry, using two anti-PGP (C219 and JSB-1) monoclonal antibodies (mAbs) and an anti-bcl-2 mAb in 115 de novo adult ALL patients. Both a longer overall survival (OS) and longer disease-free survival (DFS) were observed in PGP-negative patients (23%vs 0% at 3 years, P = 0·011 and 29%vs 0% at 2 years, P = 0·006 for C219 respectively; 42%vs 0% at 1·5 years, P = 0·004 and 53%vs 0% at 8·5 months, P = 0·00006 for JSB-1 respectively). Bcl-2 positivity was associated with a significantly higher complete remission rate (90%vs 66%, P = 0·01). Moreover, in 69 patients not presenting with either t(9;22) or B-mature immunophenotype, PGP negativity (JSB-1) maintained its significant favourable prognostic impact with regard to OS (41%vs 0% at 1·5 years, P = 0·009) and DFS (83%vs 0% at 6 months, P = 0·0005). Importantly, within a subset of 62 patients with normal (n = 31) or unknown (n = 31) karyotype, PGP (JSB-1)-negative patients showed both a significantly longer OS and DFS (63%vs 0% at 1·4 years, P = 0·018 and 84%vs 0% at 6 months, P = 0·001 respectively). In multivariate analysis, JSB-1 (P = 0·008) and cytogenetics (P = 0·02) were found to be independent prognostic factors with regard to DFS. Therefore, in adult ALL, PGP and bcl-2 represent sensitive indicators of clinical outcome, and potential targets of novel molecules aimed at overcoming chemoresistance and recurrent relapses. [ABSTRACT FROM AUTHOR]

Simple Summary: Upfront genetics/cytogenetics and minimal measurable disease (MRD) are becoming relevant biomarkers in the process of post-remission transplant allocation in AML. However, until recently the transplantation choice relied on the availability of a fully matched familiar donor, whereas individual patient- and disease-related characteristics played a secondary role in transplant allocation. In this paper we analyzed the evolution of the transplantation policy at our center in a 20-year time interval. At the beginning of our observation patients were submitted to allogeneic transplant, per protocol, mostly if a fully matched family donor was available or to autologous transplant if no fully matched family donor was identified ("donor vs. no donor" strategy) regardless of upfront genetics/cytogenetics or MRD status. Thereafter, persistence of MRD after consolidation cycle was included in the decision-making process for transplant selection. Patients with favorable and intermediate-risk cytogenetic risk were to receive allogeneic or autologous stem cell transplantation if MRD positive or negative, respectively, ("transplant vs. no transplant" strategy). In this cohort, patients with FLT3-ITD or adverse risk karyotype were submitted to allogeneic transplant as well. Measurable residual disease (MRD) is increasingly employed as a biomarker of quality of complete remission (CR) in intensively treated acute myeloid leukemia (AML) patients. We evaluated if a MRD-driven transplant policy improved outcome as compared to a policy solely relying on a familiar donor availability. High-risk patients (adverse karyotype, FLT3-ITD) received allogeneic hematopoietic cell transplant (alloHCT) whereas for intermediate and low risk ones (CBF-AML and NPM1-mutated), alloHCT or autologous SCT was delivered depending on the post-consolidation measurable residual disease (MRD) status, as assessed by flow cytometry. For comparison, we analyzed a matched historical cohort of patients in whom alloHCT was delivered based on the sole availability of a matched sibling donor. Ten-years overall and disease-free survival were longer in the MRD-driven cohort as compared to the historical cohort (47.7% vs. 28.7%, p = 0.012 and 42.0% vs. 19.5%, p = 0.0003). The favorable impact of this MRD-driven strategy was evident for the intermediate-risk category, particularly for MRD positive patients. In the low-risk category, the significantly lower CIR of the MRD-driven cohort did not translate into a survival advantage. In conclusion, a MRD-driven transplant allocation may play a better role than the one based on the simple donor availability. This approach determines a superior outcome of intermediate-risk patients whereat in low-risk ones a careful evaluation is needed for transplant allocation. [ABSTRACT FROM AUTHOR]

Myelodysplastic syndromes (MDS) are highly heterogeneous myeloid diseases, characterized by frequent genetic/chromosomal aberrations. Olaparib is a potent, orally bioavailable poly(ADP-ribose) polymerase 1 (PARP1) inhibitor with acceptable toxicity profile, designed as targeted therapy for DNA repair defective tumors. Here, we investigated olaparib activity in primary cultures of bone marrow mononuclear cells collected from patients with MDS (n = 28). A single treatment with olaparib induced cytotoxic effects in most samples, with median IC50 of 5.4 µM (2.0–24.8 µM), lower than plasma peak concentration reached in vivo. In addition, olaparib induced DNA damage as shown by a high proportion of γH2AX positive cells in samples with low IC50s. Olaparib preferentially killed myeloid cells causing a significant reduction of blasts and promyelocytes, paralleled by an increase in metamyelocytes and mature granulocytes while sparing lymphocytes that are not part of the MDS clone. Consistently, flow cytometry analysis revealed a decrease of CD117+/CD123+ immature progenitors (p < 0.001) and induction of CD11b+/CD16+ (p < 0.001) and CD10+/CD15+ (p < 0.01) neutrophils. Morphological and immunophenotypic changes were associated with a dose-dependent increase of PU.1 and CEBPA transcription factors, which are drivers of granulocytic and monocytic differentiation. Moreover, the combination of olaparib with decitabine resulted in augmented cytotoxic and differentiating effects. Our data suggest that olaparib may have therapeutic potential in MDS patients. [ABSTRACT FROM AUTHOR]

Measurable residual disease (MRD; previously termed minimal residual disease) is an independent, postdiagnosis, prognostic indicator in acute myeloid leukemia (AML) that is important for risk stratification and treatment planning, in conjunction with other well-established clinical, cytogenetic, and molecular data assessed at diagnosis. MRD can be evaluated using a variety of multiparameter flow cytometry and molecular protocols, but, to date, these approaches have not been qualitatively or quantitatively standardized, making their use in clinical practice challenging. The objective of this work was to identify key clinical and scientific issues in the measurement and application of MRD in AML, to achieve consensus on these issues, and to provide guidelines for the current and future use of MRD in clinical practice. The work was accomplished over 2 years, during 4 meetings by a specially designated MRD Working Party of the European LeukemiaNet. The group included 24 faculty with expertise in AML hematopathology, molecular diagnostics, clinical trials, and clinical medicine, from 19 institutions in Europe and the United States. [ABSTRACT FROM AUTHOR]

• The TdT enzyme may be involved in the generation of FLT3 -ITD and NPM1 mutations in AML • Median TdT expression assessed by flow cytometry on blasts is significantly higher in FLT3-ITD mutated AMLs • TdT expression levels are predictive of FLT3 -ITD mutations (C-index = 0.69) • TdT + patients had poorer OS as compared to TdT- patients in univariate analysis The terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase expressed in acute myeloid leukemias (AMLs), where it may be involved in the generation of NPM1 and FLT3 -ITD mutations. We studied the correlations between TdT expression and FLT3 -ITD or NPM1 mutations in primary AML samples, and the impact on patients' survival. TdT expression was analyzed in 143 adult AML patients by flow cytometry as percentage of positivity and mean fluorescence intensity (MFI) on blasts. TdT was positive in 49 samples (34.2%), with a median of 48% TdT-positivity (range 7-98) and a median MFI of 2.70 (range 1.23-30.54). FLT3 -ITD and NPM1 mutations were present in 24 (16.7%) and 34 (23.7%) cases, respectively. Median TdT expression on blasts was significantly higher in FLT3 -ITD+, as compared with FLT3 -ITD- AMLs (median 8% vs 0% respectively, p = 0.035). NPM1 mutational status, FLT3 -ITD allelic ratio, karyotype, and ELN risk groups, did not correlate with TdT expression or MFI on blasts. TdT + patients had poorer survival as compared to TdT-, but this result was not confirmed by the multivariable analysis, where ELN risk stratification as well as age and type of treatment remained independent prognostic factors for OS. In summary, our results support the possible implication of TdT enzyme in the generation of FLT3 -ITD mutations in AML. [ABSTRACT FROM AUTHOR]